Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol

• Published in: The Journal of biological chemistry Vol. 267; no. 10; pp. 6518 - 6525
• Main Authors: Kanematsu, T, Takeya, H, Watanabe, Y, Ozaki, S, Yoshida, M, Koga, T, Iwanaga, S, Hirata, M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 05-04-1992
• Subjects: Amino Acid Sequence; Animals; brain; Brain - metabolism; Calcium - metabolism; Calcium Channels; Cations, Divalent; Chromatography, Affinity; Cytosol - metabolism; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Hydrolysis; identification; Inositol 1,4,5-Trisphosphate Receptors; inositol 1,4,5-trisphosphate-binding protein; Inositol Phosphates - metabolism; Isoenzymes - metabolism; Molecular Sequence Data; Rats; Receptors, Cell Surface - metabolism; Receptors, Cytoplasmic and Nuclear; Substrate Specificity; Type C Phospholipases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)50458-6

In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the
second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M.,
Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When
the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution
was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration
chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins
but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase
chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C
and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations
greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations
of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa
protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence
of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.